Apoptosis In H9c2 Cells Research Articles

MiR-190-5p Alleviates Myocardial Ischemia-Reperfusion Injury by Targeting PHLPP1.

Objective Myocardial ischemia-reperfusion (I/R) injury (MIRI) refers to the more serious myocardial injury after blood flow recovery, which seriously affects the prognosis of patients with ischemic cardiomyopathy. This study explored the new targets for MIRI treatment by investigating the effects of miR-190-5p and its downstream target on the structure and function of myocardial cells. Methods We injected agomir miR-190-5p into the tail vein of rats to increase the expression of miR-190-5p in rat myocardial cells and made an I/R rat model by coronary artery occlusion. We used 2,3,5-triphenyl tetrazolium chloride staining, lactate dehydrogenase (LDH) detection, echocardiography, and hematoxylin-eosin (HE) staining to determine the degree of myocardial injury in I/R rats. In addition, we detected the expression of inflammatory factors and apoptosis-related molecules in rat serum and myocardial tissue to determine the level of inflammation and apoptosis in rat myocardium. Finally, we determined the downstream target of miR-190-5p by Targetscan system and dual luciferase reporter assay. Results The expression of miR-190-5p in an I/R rat myocardium was significantly lower than that in normal rats. After treatment of I/R rats with agomir miR-190-5p, the ischemic area of rat myocardium and the concentration of LDH decreased. The results of echocardiography and HE staining also found that overexpression of miR-190-5p improved the structure and function of rat myocardium. miR-190-5p was also found to improve the viability of H9c2 cells in vitro and reduce the level of apoptosis of H9c2 cells. The results of Targetscan system and dual luciferase reporter assay found that miR-190-5p targeted to inhibit pleckstrin homology domain leucine-rich repeat protein phosphatase 1 (PHLPP1). In addition, inhibition of PHLPP1 was found to improve the viability of H9c2 cells. Conclusion Therefore, miR-190-5p can reduce the inflammation and apoptosis of myocardium by targeting PHLPP1, thereby alleviating MIRI.

Forsythiaside A inhibits hydrogen peroxide-induced inflammation, oxidative stress, and apoptosis of cardiomyocytes

Purpose: To investigate the effect of forsythiaside A on heart failure.Methods: An in vitro cell model of myocardial injury was established by incubating H9c2 primary cardiomyocytes with hydrogen peroxide (H2O2). Apoptosis was measured by flow cytometry. Expression of inflammatory factors, including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and enzymelinkedimmunosorbent assay (ELISA). Oxidative stress was evaluated by measuring malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) levels by ELISA.Results: Incubation with H2O2 increased H9c2 cell apoptosis (p < 0.001). Treatment with forsythiaside A reduced Bax expression and enhanced Bcl-2 expression which suppressed apoptosis of H2O2- induced H9c2 cells. Forsythiaside A also attenuated the H2O2-induced increase in TNF-α and IL-6expressions in H9c2 cells (p < 0.001). The H2O2-induced increase in MDA and decrease in SOD and GSH-Px in H9c2 cells were reversed by treatment with forsythiaside A. IκBα protein expression was downregulated, whereas p65 phosphorylation (p-p65), p-IκBα, nuclear factor erythropoietin-2-related factor 2 (Nrf2), and heme oxygenase 1 (HO-1) were upregulated in H2O2-induced H9c2 cells. Forsythiaside A increased IκBα, Nrf2, and HO-1 expression and decreased p-p65 and p-IκBα expression in H2O2-induced H9c2 cells.Conclusion: Forsythiaside A exerts anti-inflammatory, anti-oxidant, and anti-apoptotic effects against H2O2-induced H9c2 cells through inactivation of NF-κB pathway and activation of Nrf2/HO-1 pathway. These results support the potential clinical application of forsythiaside A for the treatment of heart failure.

MiR-539-5p inhibits the inflammatory injury in septic H9c2 cells by regulating IRAK3

BackgroundMicroRNAs (miRNAs) have been confirmed to play a potential role in sepsis, but little is known about their role in sepsis-induced cardiomyopathy (SIC).MethodsThe model of septic cardiomyopathy was constructed with H9c2 cells induced by lipopolysaccharide (LPS), and the expression of miR-539-5p was detected by qRT-PCR assay. ELISA, CCK-8, EdU TUNEL analysis were performed to evaluate the role of miR-539-5p in inflammation response, viability, proliferation and apoptosis of LPS-treated H9c2 cells. Moreover, miRWalk and TargetScan prediction, and dual-luciferase reporter gene assays were carried out to predict and confirm the target of miR-539-5p. Furthermore, the effects of target on inflammation response, proliferation and apoptosis of LPS-induced H9c2 cells mediated by miR-539-5p was further explored.ResultsThe expression of miR-539-5p was obviously down-regulated in LPS-induced H9c2 cells. In addition, over-expression of miR-539-5p significantly inhibited the inflammation response, promoted viability and proliferation, and suppressed apoptosis of LPS-treated H9c2 cells. Moreover, interleukin-1 receptor-associated kinase 3 (IRAK3) was verified as a target of miR-539-5p by dual-luciferase reporter gene assay. Besides, IRAK3 was highly expressed in H9c2 cells transfected with miR-539-5p inhibitor detected with qRT-PCR and western blot assays. Furthermore, over-expression of IRAK3 partially weakened the effects of miR-539-5p mimic on the inflammation response, proliferation and apoptosis of LPS-induced H9c2 cells.ConclusionsMiR-539-5p potentially plays an important role in the pathogenesis of LPS-induced sepsis by targeting IRAK3, suggesting that miR-539-5p may be a potential new target for the treatment of LPS-induced sepsis.

Propofol postconditioning ameliorates hypoxia/reoxygenation induced H9c2 cell apoptosis and autophagy via upregulating forkhead transcription factors under hyperglycemia

BackgroundAdministration of propofol, an intravenous anesthetic with antioxidant property, immediately at the onset of post-ischemic reperfusion (propofol postconditioning, P-PostC) has been shown to confer cardioprotection against ischemia–reperfusion injury, while the underlying mechanism remains incompletely understood. The FoxO transcription factors are reported to play critical roles in activating cardiomyocyte survival signaling throughout the process of cellular injuries induced by oxidative stress and are also involved in hypoxic postconditioning mediated neuroprotection, however, the role of FoxO in postconditioning mediated protection in the heart and in particular in high glucose condition is unknown.MethodsRat heart-derived H9c2 cells were exposed to high glucose (HG) for 48 h (h), then subjected to hypoxia/reoxygenation (H/R, composed of 8 h of hypoxia followed by 12 h of reoxygenation) in the absence or presence of postconditioning with various concentrations of propofol (P-PostC) at the onset of reoxygenation. After having identified the optical concentration of propofol, H9c2 cells were subjected to H/R and P-PostC in the absence or presence of FoxO1 or FoxO3a gene silencing to explore their roles in P-PostC mediated protection against apoptotic and autophagic cell deaths under hyperglycemia.ResultsThe results showed that HG with or without H/R decreased cell viability, increased lactate dehydrogenase (LDH) leakage and the production of reactive oxygen species (ROS) in H9c2 cells, all of which were significantly reversed by propofol (P-PostC), especially at the concentration of 25 µmol/L (P25) (all P < 0.05, NC vs. HG; HG vs. HG + HR; HG + HR + P12.5 or HG + HR + P25 or HG + HR + P50 vs. HG + HR). Moreover, we found that propofol (P25) decreased H9c2 cells apoptosis and autophagy that were concomitant with increased FoxO1 and FoxO3a expression (all P < 0.05, HG + HR + P25 vs. HG + HR). The protective effects of propofol (P25) against H/R injury were reversed by silencing FoxO1 or FoxO3a (all P < 0.05, HG + HR + P25 vs. HG + HR + P25 + siRNA-1 or HG + HR + P25 + siRNA-5).ConclusionIt is concluded that propofol postconditioning attenuated H9c2 cardiac cells apoptosis and autophagy induced by H/R injury through upregulating FoxO1 and FoxO3a under hyperglycemia.

MiR-702-3p inhibits the inflammatory injury in septic H9c2 cells by regulating NOD1

BackgroundCardiac insufficiency is a common complication of sepsis and septic shock and is the most common cause of death in critically ill patients. Recent studies have found that microRNAs (miRNAs) play a potential role in sepsis as markers, but little is known about their functional effects on sepsis-induced cardiomyopathy (SIC). ObjectiveThis study is designed to explore the possible role and underlying mechanisms of miR-702-3p in septic cardiomyopathy. MethodsAs expected, H9c2 cells were induced with lipopolysaccharide (LPS) to construct the model of septic cardiomyopathy. The expression of miR-702-3p was detected by qRT-PCR assay and those of IL-1β, IL-6 and TNF-α by ELISA assay. The viability, proliferation and apoptosis of LPS-treated H9c2 cells were determined by CCK-8, EdU, flow cytometry and western blot assays. Moreover, Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) was predicted and confirmed as a direct target of miR-702-3p by TargetScan, miRwalk and miRDB prediction and dual-luciferase reporter gene assays. ResultsWhile LPS can weaken the viability of H9c2 cells, miR-702-3p enhances that of LPS-treated H9c2 cells by inhibit the expressions of TNF-α, IL-6, IL-1β. We found NOD1 is a target gene of miR-702-3p, and over-expression of NOD1 restores the inhibitory effects of miR-702-3p on the LPS-treated H9c2 cells. ConclusionMiR-702-3p played an important role in the pathogenesis of sepsis cardiomyopathy via targeting NOD1, suggesting that miR-702-3p may be a potential new target for the treatment of SIC.

Leflunomide inhibits inflammation and apoptosis of H9c2 cells induced by hydrogen peroxide

Purpose: To investigate the effects of leflunomide (Lef) on inflammatory response and apoptosis after myocardial infarction, and to explore its molecular mechanisms of action.Methods: H2O2 and H9c2 cells were used to establish myocardial cell injury model in vitro. H9c2 cells were divided into 3 groups: control group, H2O2 group, H2O2 + Lef group. The CCK-8 assay was used to determine the optimal concentration of H2O2 and Lef, while the expressions of TNF-α, IL-6, IL-1β, Bcl-2, Bax, Bad, TLR4, IκB-α, P65 and p-P65 were evaluated by Western blot. PCI was utilized to detect the expression of TNF-α, IL-6, IL-1β, Bcl-2, Bax and Bad mRNA. The levels of TNF-α, IL-6 and IL-1β in supernatant were assessed by ELISA, while apoptosis of the three groups was evaluated by TUNEL staining and flow cytometry.Results: Compared with H2O2 group, TNF-α, IL-6, IL-1β, Bax and Bad expressions in H2O2+Lef group were significantly reduced (p &lt; 0.05), but Bcl-2 expression significantly increased. The levels of TNF-α and IL-6 and IL-1β in supernatant of H2O2 + Lef group were also decreased compared to those in the H2O2 group (p &lt; 0.05). In addition, TUNEL-positive cells and apoptotic rates were significantly reduced after treatment with Lef. Moreover, Lef inhibited expression of TLR4 and p-P65, but activated expression of IκB-α, indicating that Lef inhibited TLR4/NF-κB pathway (p &lt; 0.05).Conclusion: The results show that Lef inhibits H2O2-induced H9c2 cell apoptosis and inflammatory responses by inhibiting TLR4/NF-κB pathway. These findings may provide new targets for the treatment of myocardial infarction.

Diazoxide Needs Mitochondrial Connexin43 to Exert Its Cytoprotective Effect in a Cellular Model of CoCl2-Induced Hypoxia.

Hypoxia is the leading cause of death in cardiomyocytes. Cells respond to oxygen deprivation by activating cytoprotective programs, such as mitochondrial connexin43 (mCx43) overexpression and the opening of mitochondrial KATP channels, aimed to reduce mitochondrial dysfunction. In this study we used an in vitro model of CoCl2-induced hypoxia to demonstrate that mCx43 and KATP channels cooperate to induce cytoprotection. CoCl2 administration induces apoptosis in H9c2 cells by increasing mitochondrial ROS production, intracellular and mitochondrial calcium overload and by inducing mitochondrial membrane depolarization. Diazoxide, an opener of KATP channels, reduces all these deleterious effects of CoCl2 only in the presence of mCx43. In fact, our results demonstrate that in the presence of radicicol, an inhibitor of Cx43 translocation to mitochondria, the cytoprotective effects of diazoxide disappear. In conclusion, these data confirm that there exists a close functional link between mCx43 and KATP channels.

Apocynum venetum leaf extract alleviated doxorubicin-induced cardiotoxicity through the AKT/Bcl-2 signaling pathway

BackgroundDoxorubicin (DOX) is a broad-spectrum anti-tumor drug that has been associated with cardiotoxicity. Plant extracts have been shown to confer protection against DOX-induced cardiotoxicity. Apocynum venetum L. belongs to the Apocynaceae family. Flavonoid extracted from Apocynum venetum L. possess various biological effects, such as lowering blood pressure levels, sedation, diuresis, anti-aging, and improving immunity. PurposeThis study investigated the mechanism by which dry leaf extract of Apocynum venetum L. (AVLE) alleviates DOX-induced cardiomyocyte apoptosis. MethodsHPLC-MS/MS and HPLC methods were used to analyze the components of AVLE. The effects of DOX and AVLE on apoptosis of H9c2 and HMC cells were assessed using the MTT assay. Calcein AM/PI, TUNEL, and flow cytometry were carried out to determine the effects of AVLE on DOX-induced apoptosis. The effect of AVLE on DOX-induced oxidative stress in cardiomyocytes was investigated using ELISA test. Mito-Tracker Red CMXRos, JC-1, and RT-qPCR assays were performed to evaluate the impact of AVLE on DOX-induced cardiomyocyte mitochondrial activity and membrane permeability. Western blot assay was carried out to determine the activation of multiple signaling molecules, including phosphorylated-protein kinase B (p-AKT), Cytochrome c, Bcl-2 family, and caspase family in the apoptosis pathway. The AKT inhibitor was used to block AKT/Bcl-2 signaling pathway to investigate the role of AKT in the protection conferred by AVLE against DOX-induced cardiotoxicity. ResultsA total of 8 compounds, including rutin, hyperoside, isoquercetin, unidentified compounds, myricetin, quercetin, quercetin-3-O-glucuronide and kaempferol, were detected in AVLE. Of note, DOX suppressed lactate dehydrogenase (LDH) levels, aggravated oxidative stress, and promoted cardiomyocyte apoptosis. It also upregulated the mRNA expression levels of voltage-dependent anion channel 1 (VDAC1), adenosine nucleotide transporter 1 (ANT1), and cyclophilin D (CYPD), while suppressing mitochondrial activity and mitochondrial membrane permeability. Treatment with DOX altered the expression levels of apoptosis-associated proteins, Bcl-2 and Bax. However, AVLE treatment alleviated DOX-induced effects on cardiomyocytes. In addition, application of AKT inhibitors promoted DOX-induced apoptosis and reversed the inhibitory effects of AVLE on DOX-induced apoptosis. ConclusionsAVLE confer cardio protection by suppressing oxidative stress and apoptosis of cardiomyocytes via AKT/Bcl-2 signaling pathway.

Cardioprotective Effects of Glucagon-like Peptide-1 (9-36) Against Oxidative Injury in H9c2 Cardiomyoblasts: Potential Role of the PI3K/Akt/NOS Pathway.

Glucagon-like peptide (GLP)-1(7-36), a major active form of GLP-1 hormone, is rapidly cleaved by dipeptidyl peptidase-4 to generate a truncated metabolite, GLP-1(9-36) which has a low affinity for GLP-1 receptor (GLP-1R). GLP-1(7-36) has been shown to have protective effects on cardiovascular system through GLP-1R-dependent pathway. Nevertheless, the cardioprotective effects of GLP-1(9-36) have not fully understood. The present study investigated the effects of GLP-1(9-36), including its underlying mechanisms against oxidative stress and apoptosis in H9c2 cells. Here, we reported that GLP-1(9-36) protects H9c2 cardiomyoblasts from hydrogen peroxide (H2O2)-induced oxidative stress by promoting the synthesis of antioxidant enzymes, glutathione peroxidase-1, catalase, and heme oxygenase-1. In addition, treatment with GLP-1(9-36) suppressed H2O2-induced apoptosis by attenuating caspase-3 activity and upregulating antiapoptotic proteins, Bcl-2 and Bcl-xL. These protective effects of GLP-1(9-36) are attenuated by blockade of PI3K-mediated Akt phosphorylation and prevention of nitric oxide synthase-induced nitric oxide production. Thus, GLP-1(9-36) represents the potential therapeutic target for prevention of oxidative stress and apoptosis in the heart via PI3K/Akt/nitric oxide synthase signaling pathway.

Long noncoding RNA MAPKAPK5-AS1 promoted lipopolysaccharide-induced inflammatory damage in the myocardium by sponging microRNA-124-3p/E2F3

BackgroundMyocardial dysfunction caused by sepsis (SIMD) leads to high mortality in critically ill patients. We investigated the function and mechanism of long non-coding RNA MAPKAPK5-AS1 (lncRNA MAPKAPK-AS1) on lipopolysaccharide (LPS)-induced inflammation response in vivo and in vitro.MethodMale SD rats were utilized for in vivo experiments. Rat cardiomyocytes (H9C2) were employed for in vitro experiments. Western blotting was employed to measure protein expression, and RT-PCR was performed to measure mRNA expression of inflammation factors. TUNEL and flow cytometry were carried out to evulate cell apoptosis.ResultThe results showed that the expression of MAPKAPK5-AS1 was increased, while the expression of miR-124-3p was decreased in the inflammatory damage induced by LPS in vivo and in vitro. Knockdown of MAPKAPK5-AS1 reduced LPS-induced cell apoptosis and inflammation response, while overexpression of miR-124-3p weakened the effects of MAPKAPK5-AS1 knockdown on LPS-induced cell apoptosis and inflammation response. Moreover, miR-124-3p was identified as a downstream miRNA of MAPKAPK5-AS1, and E2F3 was a target of miR-214-3p. MAPKAPK5-AS1 knockdown increased the expression of miR-124-3p, while miR-124-3p overexpression reduced the expression of MAPKAPK5-AS1. In addition, miR-124-3p was found to downregulate E2F3 expression in H9C2 cells.ConclusionMAPKAPK5-AS1/miR-124-3p/E2F3 axis regulates LPS-related H9C2 cell apoptosis and inflammatory response.

1-MNA Ameliorates High Fat Diet-Induced Heart Injury by Upregulating Nrf2 Expression and Inhibiting NF-κB in vivo and in vitro.

High levels of free fatty acids (FFA) are closely associated with obesity and the development of cardiovascular diseases. Recently, nicotinamide adenine dinucleotide (NAD) metabolism has emerged as a potential target for several modern diseases including diabetes. Herein, we explored the underlying mechanisms of NAD metabolism associated with the risk of cardiovascular disease. Our study found that nicotinamide N-methyltransferase (NNMT) mRNA levels were significantly increased in the hearts of FFA-bound-albumin-overloaded mice and in H9C2 cells treated with palmitic acid (PA). We studied the mechanisms underlining the anti-inflammatory and anti-oxidant activities of 1-methylnicotinamide (1-MNA), a metabolite of NNMT. We found a significantly higher level of reactive oxygen species, inflammation, apoptosis, and cell hypertrophy in PA-treated H9C2 cells and this effect was inhibited by 1-MNA treatment. in vivo, 1-MNA improved inflammation, apoptosis, and fibrosis damage in mice and this inhibition was associated with inhibited NF-κB activity. In conclusion, our study revealed that 1-MNA may prevent high fatty diet and PA-induced heart injury by regulating Nrf2 and NF-κB pathways.

Arsenic-fluoride co-exposure induced endoplasmic reticulum stress resulting in apoptosis in rat heart and H9c2 cells

Exposure to arsenic (As) or fluoride (F) has been shown to cause cardiovascular disease (CVDs). However, evidence about the effects of co-exposure to As and F on myocardium and their mechanisms remain scarce. Our aim was to fill the gap by establishing rat and H9c2 cell exposure models. We determined the effects of As and/or F exposure on the survival rate, apoptosis rate, morphology and ultrastructure of H9c2 cells; in addition, we tested the related genes and proteins of endoplasmic reticulum stress (ERS) and apoptosis in H9c2 cells and rat heart tissues. The results showed that As and/or F exposure induced early apoptosis of H9c2 cells and caused endoplasmic reticulum expansion. Additionally, the mRNA and protein expression levels of GRP78, PERK and CHOP in H9c2 cells were higher in the exposure groups than in the control group, and could be inhibited by 4-PBA. Furthermore, we found that As and/or F exposure increased the expression level of GRP78 in rat heart tissues, but interestingly, the expression level of CHOP protein was increased in the F and As groups, while significantly decreased in the co-exposure group. Overall, our results suggested that ERS-induced apoptosis was involved in the damage of myocardium by As and/or F exposure. In addition, factorial analysis results showed that As and F mainly play antagonistic roles in inducing myocardial injury, initiating ERS and apoptosis after exposure.

Protective effect of Pulsatilla saponin A on acute myocardial infarction via miR-24-3p/p16

The effect of Pulsatilla saponin A (PsA) on acute myocardial infarction (AMI) was unknown. This study targeted to examine the roles of PsA on hypoxia-triggered toxicity to H9c2 cells and reveal the potential mechanism. H9c2 cells were maintained under a hypoxic environment for 12 h to construct the AMI cell model and the cells were pretreated by PsA. Hypoxia triggered toxicity to H9c2 cells and the anti-toxicity effect of PsA was evaluated by CCK8, TUNEL, and Western blot. The levels of miR-24-3p and p16 in H9c2 cells, AMI group tissues, and their respective controls were assessed using qRT-PCR. The dual-luciferase assay was applied to verify the targeting mechanism of miR-24-3p on p16. Then the effects of miR-24-3p inhibitor or/and si-p16 on H9c2 cells treated with PsA under hypoxia were detected by CCK8, TUNEL, and Western blot. Flow cytometry was executed to determine the cell cycle. Hypoxia decreased viability and proliferation and increased apoptosis of H9c2 cells, which were ameliorated by PsA pretreatment. The level of miR-24-3p was diminished, but p16 expression was elevated in hypoxia-treated cells and AMI group tissues. MiR-24-3p could sponge p16 in hypoxia-treated cells. Furthermore, the impact of applying miR-24-3p inhibitor on PsA and hypoxia-treated cells could be reversed by si-p16. PsA relieved hypoxia-triggered cell toxicity via miR-24-3p/p16 axis. These findings provided some fresh insights into the potential therapeutic effects of the application of PsA in AMI.

Bromodomain-containing protein 7 contributes to myocardial infarction-induced myocardial injury through activating Wnt/β-catenin signaling.

Myocardial infarction (MI) is one of the most common cardiovascular diseases, inducing severe myocardial injury and leading to high mortality. Bromodomain-containing protein 7 (BRD7), a member of bromodomain-containing protein family, is involved in multiple cellular processes, such as cell cycle, transcriptional regulation, and chromatin remodeling, but the functions of BRD7 in regulating MI-associated myocardial injury are still obscure. In this work, we investigated the effect of BRD7 on MI-induced myocardial injury in vitro and in vivo. The MI model was established by ligating the left anterior descending coronary artery (LAD) of rats which were then injected with BRD7 short hairpin RNA (shRNA). The rat H9C2 cardiomyocytes were treated with hypoxia and injected with BRD7 shRNA. The expression of BRD7 in MI rat model, and hypoxia-treated H9C2 cells was detected by quantitative polymerase chain reaction (qPCR), western blot, and immunohistochemical staining. The effect of BRD7 was analyzed using western blot, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, echocardiography, and flow cytometry analysis. The expressions of Wnt/β-catenin signaling relative proteins were determined by western blot. Significantly, BRD7 was highly expressed in MI patients, MI rat models, and hypoxia treated rat H9C2 cardiomyocytes. Echocardiography analysis demonstrated that the left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) were repressed in the MI rats relative to sham group rats, while the silencing of BRD7 rescued the dysfunction in the model. We also found that BRD7 silencing reduced cardiomyocyte apoptosis in both MI rats and H9C2 cells under the treatment of hypoxia. BRD7 silencing inhibited the activation of Wnt/β-catenin signaling in H9C2 cells under the treatment of hypoxia. Moreover, Wnt agonist BML294 reversed the anti-apoptosis effect of BRD7 silencing in hypoxia-induced H9C2 cells. Collectively, we concluded that BRD7 contributed to MI-induced myocardial injury through activating Wnt/β-catenin signaling. Targeting BRD7 may become a promising therapeutic strategy for the treatment of MI-induced myocardial injury.

LncRNA RP11-400K9.4 Aggravates Cardiomyocytes Apoptosis After Hypoxia/Reperfusion Injury by Targeting miR-423.

Our study was aimed at exploring the roles of lncRNA RP11-400K9.4 (RP11-400K9.4) on hypoxia/reoxygenation (H/R) -induced cardiomyocytes apoptosis. H/R model was constructed in rat primary cardiomyocytes (PC) and H9c2 cells. In this study, the results showed that H/R significantly induced the apoptosis of PC and H9c2 cells. The expression of RP11-400K9.4 was upregulated in H/R-induced PC and H9c2 cells, but miR-423 expression was downregulated. Silencing RP11-400K9.4 could attenuate H/R-induced apoptosis in PC and H9c2 cells. We also found that miR-423 was a potential target of RP11-400K9.4. The effect of silencing RP11-400K9.4 on H/R-induced apoptosis of PC and H9c2 cells was significantly reversed by miR-423 inhibitor transfection. Furthermore, our data confirmed that silencing RP11-400K9.4 promoted the activation of phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) /extracellular signal-regulated kinase (ERK) pathways and these phenomena can be reversed by miR-423 inhibitor transfection. In conclusion, our study demonstrated that silencing RP11-400K9.4 could alleviate H/R-induced cardiomyocytes damages via suppressing apoptosis by targeting miR-423 with the activation of PI3K/AKT and MEK/ERK signaling pathways.

Study on Esculetin to reduce cardiotoxicity induced by Doxorubicin

Objective: To investigate the protective effects and mechanisms of esculetin(Esc) on H9C2 cells injury induced by doxorubicin (DOX). Methods: H9C2 cells were cultured and divided into control group, model (DOX) group and intervention (ESC + DOX) group. Flow cytometry was used to detect apoptosis of H9C2 cells and reactive oxygen (ROS) level in H9C2 cells; Western blotting to detect the cleaved Caspase-3, cleaved PARP, bid and Bcl-2 protein expression in H9C2 cells. Results: The number of apoptosis and ROS level of H9C2 cells in the model group, the expression of cleaved Caspase-3, cleaved PARP and Bid protein in the model group were obviously higher than control group; and the Bcl-2 protein expression was obviously lower (P &lt; 0.05).The number of apoptosis and ROS level, cleaved Caspase-3, cleaved PARP and Bid protein expression in H9C2 cells in intervention group were obviously lower than model group;the Bcl-2 protein expression was obviously higher (P&lt; 0.05). Conclusion: Esculetin can reduce the cardiotoxicity induced by doxorubicin by reducing apoptosis and ROS level.

Midazolam suppresses ischemia/reperfusion-induced cardiomyocyte apoptosis by inhibiting the JNK/p38 MAPK signaling pathway.

Myocardial ischemia/reperfusion (I/R) injury causes irreversible injury to the heart, thereby causing acute myocardial infarction. Midazolam is a benzodiazepine commonly utilized in anesthesia and intensive care. Research has indicated that midazolam plays a critical role in many diseases; however, the function of midazolam in myocardial injury induced by I/R still needs further investigation. The infarct size and damage to the heart tissues were examined through 2,3,5-triphenyl tetrazolium chloride (TTC) staining and hematoxylin and eosin staining. The creatine kinase-myocardial band isoenzyme, lactate dehydrogenase, and aspartate aminotransferase levels were tested using commercial kits. Cell apoptosis was determined through TUNEL staining or flow cytometry assays. Bax, Bcl-2, cleaved caspase-3, phospho-38 (p-p38), p38, p-JNK, JNK, extracellular signal-regulated kinases (ERK), and p-ERK expression was examined through Western blot. In our study, midazolam was shown to suppress the infarct size and heart tissue damage and reduce myocardial enzyme leakage in I/R rats. Additionally, midazolam was found to retard cardiomyocyte apoptosis in I/R rats. The JNK/p38 MAPK signaling pathway in I/R rats was inhibited by midazolam. Our findings demonstrated that in hypoxia/reoxygenation (H/R) - mediated H9C2 cells, anisomycin abolished the suppressive effects of midazolam on the JNK/p38 MAPK signaling pathway. Next, exploration discovered that anisomycin abolished the cytoprotective effects of midazolam on H/R-treated H9C2 cell apoptosis. In conclusion, this work demonstrated that midazolam retarded I/R-induced cardiomyocyte apoptosis by inhibiting the JNK/p38 MAPK signaling pathway. These results may provide new insight into the treatment of myocardial I/R injury.

MiR-23b targets GATA6 to down-regulate IGF-1 and promote the development of congenital heart disease

Background Congenital heart disease (CHD) is the most universal congenital defect disease. This study explores the interrelationship between miR-23b and GTAT6 in the development of CHD. Methods We collected clinical samples and constructed in vitro cell models to evaluate the expression of miR-23b, GATA6, and IGF-1. CHD cell models were constructed by hypoxia in H9C2 cells. The expression levels of GATA6 and IGF-1 in H9C2 cells were determined by western blot and qPCR. MiR-23b was knocked down by transfection miR-23b inhibitor. GATA6 knockdown or overexpression vectors were established by the lentiviral approach and cell transfection, respectively. According to the CCK-8 assay and flow cytometry analysis, the proliferation and apoptosis of H9C2 cells were detected. The binding relationship between GATA6 and miR-23b was detected by luciferase reporter assay. Results The expression level of miR-23b was escalated abnormally, while the expression levels of GATA6 and IGF-1 were decreased in the serum of CHD clinical patients and cell models. miR-23b knockdown in H9C2 cells could up-regulate the expression of GATA6, thus improved the proliferation and decreased apoptosis of H9C2 cells. Overexpression of GATA6 could up-regulate IGF-1 to promote proliferation and inhibit apoptosis in H9C2 cells. MiR-23b could target GATA6 and regulated IGF-1, thus affecting cell proliferation and apoptosis. Conclusion The expression level of miR-23b was remarkably up-regulated in serum of CHD patients and H9C2 cells in vitro, while the expression of GATA6 and IGF-1 was significantly decreased. MiR-23b could influence the proliferation and apoptosis of cardiomyocytes by targeting the down-regulation of the GATA6/IGF-1 axis.

Ononin alleviates H2O2-induced cardiomyocyte apoptosis and improves cardiac function by activating the AMPK/mTOR/autophagy pathway.

Ononin (ON) is an isoflavone with numerous reported bioactivities, including anti-oxidative, anti-inflammatory and neuroprotective effects. Autophagy is a critical homeostatic process in the body that has been reported to closely associate with the apoptotic processes of cardiomyocytes. Using flow cytometry, western blotting, echocardiography and Masson's staining, the present study investigated the effects of ON on H2O2-induced cardiomyocyte apoptosis and myocardial infarction, in addition to any potential underlying molecular mechanisms. H2O2 treatment reliably induced apoptosis in H9C2 cells. The anti-apoptotic effects of ON were revealed by flow cytometry results and by the downregulation of cleaved-caspase 3. Further investigations indicated that ON may alleviate apoptosis by enhancing autophagy, as evidenced by increased microtubule-associated proteins 1A/1B light chain 3B expression and p62 degradation. Activation of the 5' AMP-activated protein kinase (AMPK)/mTOR pathway was observed after ON administration following H2O2-induced cardiomyocyte injury. However, these anti-apoptotic effects mediated by ON were lost after autophagy inhibition by chloroquine or AMPK inhibition by Compound C. Finally, the protective effects of ON on cardiomyocytes in vitro could also be observed in vivo. A myocardial infarction model was established by ligating the left anterior descending branch of the rat heart. Using echocardiography and Masson's staining, ON was shown to increase the ejection fraction and decrease cardiac fibrosis in rats with myocardial infarction. These results suggest that ON exerts cardioprotective effects by improving autophagy via the AMPK/mTOR signaling pathway.

Protective Role of lncRNA TTN-AS1 in Sepsis-Induced Myocardial Injury Via miR-29a/E2F2 Axis.

Approximately 50% of patients with sepsis encounter myocardial injury. The mortality of septic patients with cardiac dysfunction (approx. 70%) is much higher than that of patients with sepsis only (20%). A large number of studies have suggested that lncRNA TTN-AS1 promotes cell proliferation in a variety of diseases. This study delves into the function and mechanism of TTN-AS1 in sepsis-induced myocardial injury in vitro and in vivo. LPS was used to induce sepsis in rats and H9c2 cells. Cardiac function of rats was assessed by an ultrasound system. Myocardial injury was revealed by hematoxylin-eosin (H&E) staining. Gain and loss of function of TTN-AS1, miR-29a, and E2F2 was achieved in H9c2 cellsbefore LPS treatment. The expression levels of inflammatory cytokines and cTnT were monitored by ELISA. The expression levels of cardiac enzymes as well as reactive oxygen species (ROS) activity and mitochondrial membrane potential (MMP) were measured using the colorimetric method. The expression levels of TTN-AS1, miR-29a, E2F2, and apoptosis-related proteins were measured by RT-qPCR and/or western blotting. The proliferation and apoptosis of H9c2 cells were separately detected by CCK-8 and flow cytometry. Luciferase reporter assay was used to verify the targeting relationships among TTN-AS1, miR-29a and E2F2, and RIP assay was further used to confirm the binding between miR-29a and E2F2. TTN-AS1 was lowly expressed, while miR-29a was overexpressed in the cell and animal models of sepsis. Overexpression of TTN-AS1 or silencing of miR-29a reduced the expression levels of CK, CK-MB, LDH, TNF-B, IL-1B, and IL-6 in the supernatant of LPS-induced H9c2 cells, attenuated mitochondrial ROS activity, and enhanced MMP. Consistent results were observed in septic rats injected with OE-TTN-AS1. Knockdown of TTN-AS1 or overexpression of miR-29a increased LPS-induced inflammation and injury in H9c2 cells. TTN-AS1 regulated the expression of E2F2 by targeting miR-29a. Overexpression of miR-29a or inhibition of E2F2 abrogated the suppressive effect of TTN-AS1 overexpression on myocardial injury. This study indicates TTN-AS1 attenuates sepsis-induced myocardial injury by regulating the miR-29a/E2F2 axis and sheds light on lncRNA-based treatment of sepsis-induced cardiomyopathy.